GHRP-scu-PA-32K突变体的构建、表达及纯化产物的部分性质研究  

Construction, Expression, Purification and Characterization of GHRP-scu-PA-32K

作　　者：焦建伟[1] 徐长法[1] 

机构地区：[1]北京大学蛋白质工程及植物基因工程国家重点实验室,北京100871

出　　处：《北京大学学报（自然科学版）》2000年第2期203-208,共6页Acta Scientiarum Naturalium Universitatis Pekinensis

基　　金：国家863 计划资助项目!(8631032102)

摘　　要：在ｓｃｕＰＡ３２Ｋ 的ｃＤＮＡ分子基础上经定点突变，在Ｎ 端紧接Ｌｅｕ１ 之前引入编码ＧＨＲＰ四肽的寡核苷酸序列（ＧＧＴＣＡＴＡＧＧＣＣＴ） ，构建了ＧＨＲＰｓｃｕＰＡ３２Ｋ 的突变体ｃＤＮＡ。将它克隆到表达载体ｐＣＭβｎｅｏ 中，与ｐＣＭβｄｈｆｒ 共转染ＣＨＯ／ＤＨＦＲ－ 细胞。筛选到的稳定表达株在无血清培养基的表达量为５８０ＩＵ／（１０６ 细胞·２４ ｈ） 。经锌离子螯合亲和柱纯化的产物，ＳＤＳＰＡＧＥ显示为单一蛋白条带，分子量为３３ ｋＤ，质谱法测定分子量也为３３ ｋＤ。经血纤维蛋白平板法测定比活为１５０ ０００ ＩＵ／ｍｇ 蛋白。对血纤维蛋白的亲和性，突变体为ｓｃｕＰＡ３２Ｋ 的４－５ 倍。GHRP\|scu\|PA\|32K cDNA was obtained by in vitro site\|directed\|mutagenesis to insert oligonucleotide sequence (GGTCATAGGCCT) encoding tetrapeptide Gly\|His\|Arg\|Pro into scu\|PA\|32K gene at site just preceding the amino\|terminal residue(Leu\+1). The mutant cDNA was cloned in pCM\|β\|neo expression vector and co\|transformed CHO\|DHFR\+- cells together with pCM\|dhfr.The stable expression cell line was selected. Expression level of the line cultured in serum\|free medium was 580?IU/10\+6 cell/24?h.The product expressed was purified by Zn\+\{2+\}\|Sepharose affinity chromatography. The apparent molecular weight of purified GHRP\|scu\|PA\|32K was 33?kD by SDS\|PAGE and mass spectrometry and its specific activity was 15×10\+4?IU/mg protein, according to fibrin plate determination. The affinity for fibrin of GHRP\|scu\|PA\|32K was 4 5 times higher than that of scu\|PA\|32K.

关 键 词：GHRP四肽 定点突变 溶栓药物 scu-PA-32K 突变体 

分 类 号：R94[医药卫生—药剂学]